人高亲和力IgE受体α链基因克隆及表达  

The gene cloning and expression of human high-affinity IgE receptor alpha-chain

作　　者：孙蔚凌[1] 毕志刚[1] 

机构地区：[1]南京医科大学第一附属医院皮肤科,南京210029

出　　处：《中国麻风皮肤病杂志》2005年第8期609-612,共4页China Journal of Leprosy and Skin Diseases

摘　　要：目的:克隆人高亲和力IgE受体α链基因,并进行原核表达,制备出可溶性FcεRIα蛋白。方法:应用RT-PCR技术,从皮肤组织的总RNA中扩增人高亲和力IgE受体α链基因,连接至PUCm-T载体,重组克隆进行DNA测序。将测序证实的目的片段与高效表达载体pET30a连接,构建重组质粒并转入表达宿主菌BL21(DE3),以IPTG诱导表达,Ni-NTA树脂柱亲和层析纯化。结果:RT-PCR扩增出一个约950bp的DNA片段,测序结果显示该片段与GenBank上登录的FcεRIα基因序列完全一致;经原核表达后,获得相对分子质量37000的FcεRIα融合蛋白。结论:本实验成功克隆、表达了人FcεRIα基因,制备出可溶性FcεRIα蛋白,为抗FcεRIα抗体的检测及自身免疫性慢性荨麻疹发病机制的研究提供了条件。Objective： To clone and to express human high - affinity IgE receptor alpha - chain gene. and to obtain a recombinant preparation of the soluble protein of FcεRlα. Methods： The cDNA of FcεRlα was obtained by reverse transeription and amplified, and then linked with pUCm - T vector and analyzed by automated sequencing. The targeted DNA fragments were subcloned into expression veetor pFT30a and transformed into the competent E. coil BL（DE3）, and induced by IPTG for the recombinant protein expressions. The filsion protein was purified by Ni- NTA adsorption chromatography and characterized by SDS- PAGE. Results： A 950bp cDNA was amplified by RT- PCR and the clone was FcεRlα exactly. A fusion prntein with molecular weight of 37 000 was expressed as expoected. Conclusion： Success in cloning and expression gene of FcεRlα and preparation the fusion protein of FcεRlα has provided a basis for the detection of anti - FcεRlα auto - antibodies and for the study of the pathogenesis of autoimmune chronic urticaria.

关 键 词：基因克隆 基因表达 人高亲和力IgE受体α链 自身免疫性慢性荨麻疹 高亲和力IGE受体 α链基因 BL21(DE3) DNA测序 DNA片段 高效表达载体 

分 类 号：R758.24[医药卫生—皮肤病学与性病学]